Project Summary/Abstract The broad goal of this proposal is to understand how memories are formed and stored in the brain. Pavlovian fear conditioning has proven to be a useful paradigm in elucidating the molecular mechanisms underlying memory formation and storage in cells. Indeed, good evidence now exists suggesting that memories formed using this paradigm require dynamic post-translational modification of proteins in neurons. Recently, evidence has emerged demonstrating that protein ubiquitination is critically involved in memory formation in the brain, primarily through targeting proteins for degradation by the proteasome. However, much remains unknown about how other ubiquitin marks which are independent of the proteasome are involved in the memory storage process. In our preliminary studies, we found that in the amygdala fear learning increased the levels of linear ubiquitinated proteins, an atypical ubiquitin modification that is not targeted for degradation by the proteasome complex. Importantly, these increases occurred selectively in the nucleus, but not the cytoplasm or at synapses, and targeted the transcription factor p65, suggesting that linear ubiquitination may be involved in transcriptional control during memory formation. The work in this proposal is designed to answer important questions about whether linear ubiquitination is involved in gene transcription critical for fear memory formation in the amygdala. Using a combination of protein purification methods and mass spectrometry, Aim 1 will identify what proteins are targeted by linear ubiquitination following learning. Additionally, this aim will correlate these identified proteins with next generation RNA-seq data obtained following siRNA-mediated reductions in linear ubiquitination within the amygdala, which will allow an unbiased, whole genome analysis of how this ubiquitin mark is involved in transcriptional processes during the process of memory formation. Aim 2 directly tests the functional role of linear ubiquitination in memory formation by reducing or enhancing this ubiquitin mark in the amygdala via in vivo siRNA or CRISPR-dCas9 manipulations, respectively, and testing memory retention for a contextual fear conditioning task. Collectively, this research will provide critical information regarding how linear ubiquitination is involved in transcriptional regulation during memory formation and whether it is critical for learning-dependent synaptic plasticity. This could provide potentially useful information on how memories are stored in the brain which could have important implications for the treatment of memory impairments associated with a variety of psychiatric disorders.